The baculovirus expression vector system (BEVS) has been successfully utilized to produce thousands of proteins for use as vaccines, therapeutics, and for structure-function studies. One limitation of BEVS is the propensity of baculoviruses to accumulate transposon insertions into the fp25k gene leading to the few polyhedra (FP) phenotype. This mutation shifts the balance of virus production from occlusion-derived viruses, which are not infectious in tissue culture, to budded viruses, which are the form of virus that is used in baculovirus expression. These higher levels of budded virus would be advantageous for BEVS users, but FP mutants are also deficient in transcription from the polyhedrin promoter, which drives expression of target genes. Baculoviruses also rapidly accumulate defective interfering particles (DIP), which are linked to a sharp decrease in target gene expression due to deletion of the target gene and/or viral genes needed for its expression. One factor that promotes DIP formation is transposition into fp25k. The goal of this project is to develop baculovirus expression vectors that allow for manipulation of the fp25k gene. By reducing or eliminating expression of FP25K during virus amplification, we expect to obtain 5- to10- fold higher levels of budded virus titers because most of the replicative potential of the cel would go to producing budded virus instead of occlusion-derived virus. Then FP25K expression would be restored when target protein expression is desired. This would significantly lower costs for large-scale production of baculovirus-expressed proteins because high titer BV stocks would be easier to produce and the occurrence of deleterious mutations would be reduced. ParaTechs will pursue two complementary approaches to achieve this goal. One involves the production of a virus with an inducible fp25k gene that can be turned off during amplification and activated during target protein expression. The other approach utilizes a virus with a deletion in fp25k coupled with a cell line that expresses FP25K. Each of these approaches has advantages and disadvantages, both in the design phase and for the end user. Experiments described in this proposal will determine which provides higher levels of BV production, polyhedrin-linked expression, and stable genome maintenance. Ultimately, the system developed here would be combined with ParaTechs vankyrin expression technology, which increases polyhedrin-driven expression in BEVS from 2- to 20-fold.